Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

Abstract An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Low cost single-step purification of endoglucanase from Aspergillus fumigatus ABK-9.

Low cost agro-waste was used as adsorption support for single-step purification of endoglucanase from the culture filtrate of A. fumigatus ABK-9. Among various agro-waste substrates, 1% NaOH pretreated rice bran was proved to be the best for adsorbing about 74.8 and 71.1% of endoglucanase at 4 °C and 10 °C respectively. Langmuir type adsorption isotherm at 4 °C showed maximum adsorption of enzyme at pH 5.0, which was in the range of optimum pH of the enzyme. The rice bran column bound enzyme was maximally eluted by a mixture of acetate buffer (0.05 M, pH 5.5) and ethanol (40%, v/v) at a ratio of 3:2 and a flow rate of 1 mL/min. A 5.52-fold purification of the enzyme was achieved from culture supernatant. The specific activity and recovery yield after purification were 294.0 U/mg and 40.15%, respectively, which were comparable with other contemporary protocols. The homogeneity of the enzyme was tested through sodium dodecyl sulphate polyacrylamide gel electrophoresis and a single band of 56.3 kDa was observed. Zymogram analysis finally confirmed the occurrence of endoglucanase in the single band.

Production and partial characterization of serine and metallo peptidases secreted by Aspergillus fumigatus Fresenius in submerged and solid state fermentation

Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 ºC with an inoculum of 1 x 10(6) spores and yielded 1500 active units (UµmL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 ºC and yielded 40 UµmL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 ºC, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase.

Expression of a heat stable phytase from Aspergillus fumigatus in tobacco (Nicotiana tabacum L. cv. NC89).

Aspergillus fumigatus contains a heat-stable phytase of great potential. To determine whether this phytase could be expressed in plants as a functional enzyme, we introduced the phytase gene from A. fumigatus (fphyA) in tobacco (Nicotiana tabacum L. cv. NC89) by Agrobacterium-mediated transformation. Phytase expression was controlled by the cauliflower mosaic virus (CaMV) 35S promoter. Secretion of recombinant phytase (tfphyA) to the extracellular fluid was established by use of the signal sequence from tobacco calreticulin. Forty-one independent transgenic plants were generated. Single-copy line A was selected based on segregation of T1 seeds for kanamycin resistance, phytase expression and Southern blotting analysis for use in further study. After 4-weeks of plant growth, the phytase was accumulated in leaves up to 2.3% of total soluble protein. tfphyA was functional and shared similar profiles of pH, temperature and thermal stability to the same enzyme expressed in Pichia pastoris (pfphyA). The expressed enzyme had an apparent molecular mass of 63 kDa and showed maximum activity at pH 5.5, and temperature, 55 degrees C. It had a high thermostability and retained 28.7% of the initial activity even after incubation at 90 degrees C for 15 min. The above results showed that the thermostable A. fumigatus phytase could be expressed in tobacco as a functional enzyme and thus has the potential of overexpressing it in other crop plants also.

Purification and characterization of a low molecular weight xylanase from solid-state cultures of Aspergillus fumigatus fresenius

A xylan-degrading enzyme (xylanase II) was purified to apparent homogeneity from solid-state cultures of Aspergillus fumigatus Fresenius. The molecular weight of xylanase II was found to be 19 and 8.5 kDa, as estimated by SDS-PAGE and gel filtration on FPLC, respectively. The purified enzyme was most active at 55 (degree)C and pH 5.5. It was specific to xylan. The apparent Km and Vmax values on soluble and indisoluble xylans from oat spelt and birchwood showed that xylanase II was most active on soluble birchwood xylan. Studies on hydrolysis products of various xylans and xylooligormers by xylanase II on HPLC showed that the enzyme released a range of products from xylobiose to xylohexaose, with a small amount of xylose from xylooligomers, and presented transferase activity.

n-Propanol as a substrate for assaying the ligninperoxidase activity of Phanerochaete chrysoporium.

The steady state kinetics of ligninperoxidase catalysed reaction using n-propanol as the organic substrate and monitoring the formation of propanaldehyde at lambda = 300 nm spectrophotometerically as functions of different reaction parameters has been studied. It has been concluded that n-propanol can be used as a substrate for analysing the activity of ligninperoxidase. The turnover number of ligninperoxidase of Phanerochaete chrysosporium using n-propanol as substrate has been found to be higher approximately by a factor of 10(3) as compared to that using veratryl alcohol as the substrate. The method works in assaying the activity of ligninperoxidase produced by Aspergillus fumigatus indicating that it can be used for assaying the ligninperoxidase activities produced by other microorganisms also and is not limited to assaying the ligninase activity produced by Phanerochaete chrysosporium alone. Under identical experimental conditions, horseradish peroxidase does not show peroxidase activity using n-propanol as substrate indicating that the method does not interfere with the activities of other peroxidases.

An extracellular phosphatase from Aspergillus fumigatus.

A potential producer of extracellular phosphatase has been isolated and identified as A. fumigatus. The fungal phosphatase is active in pH range 5 to 8 and its temperature optimum is 65 degrees C. The mineralisation of organic phosphates present in Neem cake and press mud by this enzyme has been demonstrated.

Production of serine chymotrypsin-like elastate by aspergillus fumigatus strains

Treinta y cuatro cepas de Aspergillus fumigatus aisladas del aire, crín de caballo, suelo agrícola y del hombre, fueron examinadas con el fin de evaluar la producción de elastasa. Las cepas de Aspergillus fumigatus fueron cultivadas en un medio sólido con elastina, apreciándose en ella su amplio solubilización por la acción del hongo. Los aislamientos fúngicos provenientes de muestras aisladas del hobre y de suelos agrícolas fueron detectados como los más altos productores de elastasa. Ocho de las 34 cepas fueron desarrollas en 4 diferentes medios líquidos en las cuales se investigó la actividad proteolítica total y específica. Los resultados de este experimento sugieren que la producción de elastasa es inducida por la presencia de elastina como sustrato y que la primera es una enzima semejante a la quimiotripsina. El perfil inhibitorio comprobó que la elastina de A.fumigatus, es una serina-proteinasa